Antibacterial agents

ABSTRACT

The invention provides an antibacterial compound of formula (I) or a salt thereof, as well as an antibacterial compound of formula (II) or a salt thereof, wherein R1, R2, X, Y and n have any of the values defined in the specification.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority from U.S. Provisional Application No.62/012,850, filed Jun. 16, 2014, which application is hereinincorporated by reference.

GOVERNMENT FUNDING

This invention was made with government support under R01 HL107913awarded by the National Institutes of Health and under P200A120078awarded by the U.S. Department of Education fellowship for GraduateAssistance in Areas of National Need. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The development of antibiotic-resistant bacteria is a prevalent concernthat has prompted the development of new antimicrobial agents (Park, etal., International Journal of Molecular Sciences 2011, 12, 5971;Laverty, et al., International Journal of Molecular Sciences 2011, 12,6566; Tew, et al., Accounts of chemical research 2010, 43, 30; Boucher,et al., Clinical Infectious Diseases 2009, 48, 1). As an alternative toconventional antibiotics, antimicrobial peptides (AMPs) have receivedwidespread attention. Many of the naturally occurring AMPs elicitantibacterial activity by targeting the cellular membrane (Park, et al.,International Journal of Molecular Sciences 2011, 12, 5971; Laverty, etal., International Journal of Molecular Sciences 2011, 12, 6566; Tew, etal., Accounts of chemical research 2010, 43, 30). Although thesepeptides have diverse primary structures, many exhibit a net cationiccharge and facially amphiphilic secondary structure in which hydrophobicand hydrophilic domains exist on opposite ‘faces’ of the molecule (Zhao,Y. Current Opinion in Colloid & Interface Science 2007, 12, 92); it isthe cationic, amphiphilic character that appears to give rise to AMPs'unique mechanism of action (Park, et al., International Journal ofMolecular Sciences 2011, 12, 5971; Laverty, et al., InternationalJournal of Molecular Sciences 2011, 12, 6566; Tew, et al., Accounts ofchemical research 2010, 43, 30). These AMPs first interact withnegatively charged bacterial membranes via electrostatic bonding(Laverty, G.; Gorman, S. P.; Gilmore, B. F. International Journal ofMolecular Sciences 2011, 12, 6566; Brogden, K. A. Nature ReviewsMicrobiology 2005, 3, 238). After the initial interaction, AMPs'hydrophobic domains interact with the hydrophobic membrane interior,ultimately disrupting the membrane and resulting in cell death (Laverty,G.; Gorman, S. P.; Gilmore, B. F. International Journal of MolecularSciences 2011, 12, 6566; Brogden, K. A. Nature Reviews Microbiology2005, 3, 238). Owing to their membrane-targeting activity, AMPS exhibitreduced instances of bacterial resistance and are promising antibioticalternatives (Laverty, G.; Gorman, S. P.; Gilmore, B. F. InternationalJournal of Molecular Sciences 2011, 12, 6566; Grenier, et al.,Bioorganic & Medicinal Chemistry Letters 2012, 22, 4055; Ling, et al.,Nature 2015, advance online publication). However, high production costsand instability in the presence of proteases, has limited their clinicalapplication (Tew, et al., Accounts of chemical research 2010, 43, 30;Scorciapino, et al., Biophys. J. 2012, 102, 1039; Hancock, R. E. W.;Sahl, H.-G. Nature Biotechnology 2006, 24, 1551).

Accordingly, there is a need for new agents that have antibacterialproperties.

SUMMARY OF THE INVENTION

Applicant has discovered novel cationic amphiphiles, which may be usedas therapeutic compounds. These amphiphiles may also be useful inbiomedical applications, including antimicrobial and deliveryapplications. Research suggests that such cationic amphiphiles can selfassemble into micelles, complex with liposomes, or be formulated intonanoparticles for various delivery applications. These amphiphiles couldthus be used as therapeutic compounds, as delivery vehicles forbioactives, including oligonucleotides, or for diagnostics.Additionally, these cationic amphiphiles could improve the performanceof current antimicrobial products. Thus, therapeutic compounds aredescribed below. In particular, compounds with antibacterial propertiesare described below.

Accordingly, the invention provides a compound of formula I:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is optionally substituted withone or more NR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is optionally substituted withone or more NR_(a)R_(b);

each X is independently (C₁-C₂₀)alkyl;

R_(a) and R_(b) are each independently H or (C₁-C₆)alkyl;

each Y is independently —NH₂, —N⁺(R^(c))₃W⁻, —NH—C(═NH)—NH₂ or —NH—BOC;

each R^(c) is independently (C₁-C₆)alkyl;

W is a counter ion; and

n is 1, 2, 3, 4, 5, 6, 7, or 8;

or a salt thereof.

The invention also provides a compound of formula II:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

each X is independently (C₁-C₂₀)alkyl;

each R_(a) is independently H or (C₁-C₆)alkyl;

each R_(b) is independently H, (C₁-C₆)alkyl or —C(═NH)NH₂; and

n is 1, 2, 3, 4, 5, 6, 7, or 8;

or a salt thereof.

The invention also provides a method for treating a bacterial infectionin a mammal comprising administering to the mammal an effective amountof a compound of formula I, or a pharmaceutically acceptable saltthereof, or a compound of formula II, or a pharmaceutically acceptablesalt thereof.

The invention also provides a composition comprising a compound offormula I, or a pharmaceutically acceptable salt thereof, or a compoundof formula II, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable vehicle.

The invention also provides a compound of formula I, pharmaceuticallyacceptable salt thereof, or a compound of formula II, or apharmaceutically acceptable salt thereof, for the prophylactic ortherapeutic treatment of a bacterial infection.

The invention also provides a compound of formula I, pharmaceuticallyacceptable salt thereof, or a compound of formula II, or apharmaceutically acceptable salt thereof, for use in medical treatment.

The invention also provides the use of a compound of formula I, or apharmaceutically acceptable salt thereof, or a compound of formula II,or a pharmaceutically acceptable salt thereof, for the preparation of amedicament for treating a bacterial infection in a mammal.

The invention also provides processes and intermediates disclosed hereinthat are useful for preparing compounds of formula I, or salts thereof,or compounds of formula II, or salts thereof.

As described in the Examples, experiments measuring the minimuminhibitory concentrations (MIC) and minimum bactericidal concentrations(MBC) against gram positive (S. aureus) and gram negative (E. coli)bacteria were performed using compounds of formula (I) and (II), withcertain compounds showing activities comparable to conventionalantibiotics. The compounds may be useful as therapeutic agents forclinical applications. The compounds may also be useful to provide anantibiotic effect in soaps and coatings or as delivery agents for othertherapeutic or diagnostic agents. The compounds may kill bacterial cellsthrough a membrane disrupting mechanism, which is difficult for bacteriato develop resistance against. Additionally, the compounds are simpleand inexpensive to prepare.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A representative series of proposed cationic amphiphiles, havinga tartaric acid-based backbone, that can be explored for antimicrobialapplications. Amphiphiles of different hydrophobicities and differentamine-moieties will be investigated. Examples of PEGylation strategiesare presented for one amphiphile structure.

FIG. 2. Synthetic scheme used to synthesize tartaric acid-basedamphiphiles containing primary amine-terminated aliphatic chains.

FIG. 3. Proton nuclear magnetic resonance spectra for synthesis of atartaric-acid based amphiphile with primary amine-terminated aliphaticarms.

FIG. 4. Proposed interaction between bola-like amphiphiles andgram-positive bacteria (top panel) and gemini-like amphiphiles andgram-negative bacteria (bottom panel).

FIG. 5. Representations of amphiphile classes previously investigatedfor antimicrobial applications (A); Chemical structures andrepresentations of bola-like (left) and gemini-like (right) amphiphilesinvestigated herein (B).

FIG. 6. Raw Langmuir monolayer data depicting the surface pressureincrease upon injection of B11 (panel A) or G7 (panel B) into theaqueous subphase of a trough containing DOPC (1), DOPG (2), or DOPC:DOPG(1:1 mol ratio, (3)) monolayers at initial surface pressures ofapproximately 26 mN/m.

FIG. 7. Interaction of B11 (triangles, A) and G7 (diamonds, B) with DOPG(solid) or DOPC:DOPG (1:1 mol ratio, open) lipid monolayers indicated bychange in surface pressure as a function of initial surface pressure.

FIG. 8. ITC traces obtained from titrating DOPC:DOPG (1:1 mol ratio)into B11 (A) and G7 (B). Upper curves depict heat flow as a function oftime, whereas lower curves depict the corresponding integrated area ofeach peak as a function of injection number.

FIG. 9. Antimicrobial screening of bola-like amphiphiles B7 (top), B9(middle), and B11 (bottom) against E. coli (left) and S. aureus (right)as determined by a disk diffusion assay. Zones of inhibition (i.e., nobacterial growth) correspond to antimicrobial activity.

FIG. 10. Antimicrobial screening of gemini-like amphiphiles G7 (top), G9(middle), and G11 (bottom) against E. coli (left) and S. aureus (right)as determined by a disk diffusion assay. Zones of inhibition (i.e., nobacterial growth) correspond to antimicrobial activity.

FIG. 11. Langmuir monolayer data depicting surface pressure increaseupon injection of B11 (panel A) or G7 (panel B) into the aqueoussubphase of a trough containing either DOPC (1) or DOPG (2) lipidmonolayers at initial surface pressures of approximately 25 mN/m.

FIG. 12. ITC traces obtained from titrating DOPC into B11 (panel A) andG7 (panel B). Upper curves depict heat flow as a function of time,whereas lower curves depict the corresponding integrated area of eachpeak as a function of injection number. Heat flow was negligible forboth titrations.

FIG. 13. A. Structure of G5 amphiphile; and B. Structures of G7derivatives.

DETAILED DESCRIPTION

Compounds of Formula (I)

Accordingly, the invention provides a compound of formula I:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is optionally substituted withone or more NR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is optionally substituted withone or more NR_(a)R_(b);

each X is independently (C₁-C₂₀)alkyl;

R_(a) and R_(b) are each independently H or (C₁-C₆)alkyl;

each Y is independently —NH₂, —N⁺(R^(c))₃W⁻, —NH—C(═NH)—NH₂ or —NH—BOC;

each R^(c) is independently (C₁-C₆)alkyl;

W is a counter ion; and

n is 1, 2, 3, 4, 5, 6, 7, or 8;

or a salt thereof.

In certain embodiments, each Y is independently —NH₂.

In certain embodiments, each Y is independently —N⁺(R^(c))₃W⁻.

In certain embodiments, each Y is independently —NH—C(═NH)—NH₂.

In certain embodiments, each Y is independently —NH—BOC.

In certain embodiments, each R_(a) is independently H. In certainembodiments, each R_(a) is independently (C₁-C₆)alkyl.

In certain embodiments, each R_(b) is independently H. In certainembodiments, each R_(b) is independently (C₁-C₆)alkyl.

In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4. In certain embodiments, n is 5.

In certain embodiments, a compound of formula I is a compound of formulaIc:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is optionally substituted withone or more NR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is optionally substituted withone or more NR_(a)R_(b);

each X is independently (C₁-C₂₀)alkyl;

R_(a) and R_(b) are each independently H or (C₁-C₆)alkyl;

each Y is independently —NH₂, —N⁺(R^(c))₃W⁻, —NH—C(═NH)—NH₂ or —NH—BOC;

each R^(c) is independently (C₁-C₆)alkyl;

W is a counter ion; and

n is 1, 2, 3, 4, 5, 6, 7, or 8;

or a salt thereof.

In certain embodiments, a compound of formula I is a compound of formulaIb:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl; and

R² is a polyether or a (C₁-C₆)alkyl;

or a salt thereof.

In certain embodiments, a compound of formula I is a compound of formulaIb′:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl; and

R² is a polyether or a (C₁-C₆)alkyl;

or a salt thereof.

In certain embodiments, a compound of formula I is a compound of formulaIa:

or a salt thereof.

In certain embodiments, a compound of the invention is selected from:

and salts thereof.

In certain embodiments, a compound of the invention is selected from:

Compounds of Formula (II)

The invention also provides a compound of formula II:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

each X is independently (C₁-C₂₀)alkyl;

each R_(a) is independently H or (C₁-C₆)alkyl;

each R_(b) is independently H, (C₁-C₆)alkyl or —C(═NH)NH₂; and

n is 1, 2, 3, 4, 5, 6, 7, or 8;

or a salt thereof.

In certain embodiments, each R_(a) is independently H. In certainembodiments, each R_(a) is independently (C₁-C₆)alkyl.

In certain embodiments, each R_(b) is independently H. In certainembodiments, each R_(b) is independently (C₁-C₆)alkyl. In certainembodiments, each R_(b) is independently —C(═NH)NH₂.

In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4. In certain embodiments, n is 5.

In certain embodiments, a compound of formula II is a compound offormula IIa:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

each X is independently (C₁-C₂₀)alkyl;

R_(a) is each independently H or (C₁-C₆)alkyl;

R_(b) is each independently H, (C₁-C₆)alkyl or —C(═NH)NH₂; and

n is 1, 2, 3, 4, 5, 6, 7, or 8;

or a salt thereof.

In certain embodiments, a compound of formula II is a compound offormula IIb:

wherein:

R¹ is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

R² is a polyether or a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b);

each R_(a) is H; and

each R_(b) is H;

or a salt thereof.

In certain embodiments, a compound of formula II is a compound offormula IIc:

or a salt thereof.

In certain embodiments, a compound of the invention is selected from:

and salts thereof.

In certain embodiments, a compound of the invention is selected from:

Variables “R¹” and “R²”

As described herein, certain embodiments of the invention providecompounds of formula I, wherein R¹ is a polyether or a (C₁-C₆)alkyl thatis optionally substituted with one or more NR_(a)R_(b); and R² is apolyether or a (C₁-C₆)alkyl that is optionally substituted with one ormore NR_(a)R_(b). Certain embodiments of the invention also providecompounds of formula II, wherein R¹ is a polyether or a (C₁-C₆)alkylthat is substituted with one or more NR_(a)R_(b); and R² is a polyetheror a (C₁-C₆)alkyl that is substituted with one or more NR_(a)R_(b).

In certain embodiments, R¹ is a (C₁-C₆)alkyl that is optionallysubstituted with one or more NR_(a)R_(b).

In certain embodiments, R¹ is a (C₁-C₆)alkyl that is substituted withone or more NR_(a)R_(b).

In certain embodiments, R¹ is methyl, ethyl, propyl, isopropyl, butyl,isobutyl, or sec-butyl, optionally substituted with one or moreNR_(a)R_(b).

In certain embodiments, R¹ is methyl, ethyl, propyl, isopropyl, butyl,isobutyl, or sec-butyl.

In certain embodiments, R¹ is propyl.

In certain embodiments, R¹ is a polyether. As used herein, the termpolyether includes poly(alkylene oxides) having between about 2 andabout 150 repeating units. Typically, the poly(alkylene oxides) havebetween about 50 and about 115 repeating units. The alkylene oxide unitscontain from 2 to 10 carbon atoms and may be straight chained orbranched. Preferably, the alkylene oxide units contain from 2 to 10carbon atoms. Poly(ethylene glycol) (PEG) is a specific example of apoly(alkylene oxide). Alkoxy-, amino-, carboxy-, and sulfo-terminatedpoly(alkylene oxides) are also examples, with methoxy-terminatedpoly(alkylene oxides) being a specific example.

In one embodiment the polyether has the following structure:R₅—(R₆—O—)_(a)—R₆—

wherein R₅ is a 1 to 20 carbon straight-chain or branched alkyl group,—OH, —OR₇, —NH₂, —NHR₇, —NHR₇R₈, —CO₂H, —SO₃H (sulfo), —CH₂—OH,—CH₂—OR₇, —CH₂—O—CH₂—R₇, —CH₂—NH₂, —CH₂—NHR₇, —CH₂—NR₇R₈, —CH₂CO₂H,—CH₂SO₃H, or —O—C(═O)—CH₂—CH₂—C(═O)—O—;

R₆ is a 1 to 10 carbon straight-chain or branched divalent alkylenegroup;

each R₇ and R₈ is independently a 1 to 6 carbon straight-chain orbranched alkylene group; and

a is an integer from 2 to 150, inclusive.

In certain embodiments, a is an integer from 20 to 140, inclusive. Incertain embodiments, a is an integer from 50 to 130, inclusive. Incertain embodiments, a is an integer from 75 to 130, inclusive. Incertain embodiments, a is an integer from 100 to 130, inclusive. Incertain embodiments, a is 113.

In another embodiment the polyether is methoxy terminated poly(ethyleneglycol).

In certain embodiments, R² is a (C₁-C₆)alkyl that is optionallysubstituted with one or more NR_(a)R_(b).

In certain embodiments, R² is a (C₁-C₆)alkyl that is substituted withone or more NR_(a)R_(b).

In certain embodiments, R² is methyl, ethyl, propyl, isopropyl, butyl,isobutyl, or sec-butyl, optionally substituted with one or moreNR_(a)R_(b).

In certain embodiments, R² is methyl, ethyl, propyl, isopropyl, butyl,isobutyl, or sec-butyl.

In certain embodiments, R² is propyl.

In certain embodiments, R² is a polyether.

Variable “X”

As described herein, in certain embodiments of compounds of formula Iand II, each X is independently (C₁-C₂₀)alkyl. In certain embodiments,each X is independently (C₂-C₂₀)alkyl. In certain embodiments, each X isindependently (C₄-C₁₂)alkyl. In certain embodiments, each X isindependently (C₆)alkyl. In certain embodiments, each X is independently(C_(T))alkyl. In certain embodiments, each X is independently (C₈)alkyl.In certain embodiments, each X is independently (C₉)alkyl. In certainembodiments, each X is independently (C₁₀)alkyl. In certain embodiments,each X is independently (C₁₁)alkyl. In certain embodiments, each X isindependently (C₁₂)alkyl.

Compositions

As described herein, compounds of the invention may be formulated ascompositions. Accordingly, certain embodiments of the invention providea pharmaceutical composition comprising a compound of formula I, or apharmaceutically acceptable salt thereof, or a compound of formula II,or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Certain other embodiments provide a soap comprising a compound offormula I, or a salt thereof, or a compound of formula II, or a saltthereof.

Certain embodiments of the invention provide a coating or paintcomprising a compound of formula I, or a salt thereof, or a compound offormula II, or a salt thereof.

Certain embodiments of the invention provide composition comprising acompound of formula I, or a salt thereof, or a compound of formula II,or a salt thereof, and a therapeutic or diagnostic agent.

Methods of Use

Certain embodiments of the invention provide a method for treating abacterial infection in a mammal comprising administering to the mammalan effective amount of a compound as described herein, or apharmaceutically acceptable salt thereof.

In certain embodiments, the bacterial infection is a Gram-negativebacterial strain infection.

In certain embodiments, the Gram-negative bacterial strain is selectedfrom the group consisting of Escherchia coli, Caulobacter crescentus,Pseudomonas aeruginosa, Agrobacterium tumefaciens, Branhamellacatarrhalis, Citrobacter diversus, Enterobacter aerogenes, Enterobactercloacae, Enterobacter sakazakii, Enterobacter asburiae, Pantoeaagglomerans, Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiellarhinoscleromatis, Proteus mirabilis, Salmonella typhimurium, Salmonellaenteriditis, Serratia marcescens, Shigella sonnei, Neisseriagonorrhoeae, Acinetobacter baumannii, Acinetobacter calcoaceticus,Acinetobacter lwoffi, Salmonella enteriditis, Fusobacterium nucleatum,Veillonella parvula, Bacteroides forsythus, Actinobacillusactinomycetemcomitans, Aggregatibacter actinomycetemcomitans,Porphyromonas gingivalis, Helicobacter pylori, Francisella tularensis,Yersinia pestis, Borrelia burgdorferi, Neisseria meningitides,Burkholderia cepacia, Brucella neotomae, Legionella pneumophila, Ypseudotuberculosis, and Haemophilus influenzae.

In certain embodiments, the bacterial infection is a Gram-negativebacterial strain infection and a compound of formula II is administeredto the mammal.

In certain embodiments, the bacterial infection is a Gram-positivebacterial strain infection.

In certain embodiments, the Gram-positive bacterial strain is selectedfrom the group consisting of Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Streptococcus pyogenes,Streptococcus faecalis, Enterococcus faecalis, Enterococcus faecium,Bacillus subtilis, Micrococcus luteus, Mycobacterium tuberculosis,Bacillus anthracis, Bacillus cereus, Clostridium difficile,Propionibacterium acnes, Streptococcus mutans, Actinomyces viscosus,Actinomyces naeslundii, Streptococcus sanguis, Streptococcus pneumoniae,Listeria monocytogenes and Streptococcus salivarius.

In certain embodiments, the bacterial infection is a Gram-positivebacterial strain infection and a compound of formula I is administeredto the mammal.

In certain embodiments, the bacterial infection is a Gram-positivebacterial strain infection and a compound of formula II is administeredto the mammal.

In certain embodiments, the Gram-positive bacterial strain isMycobacterium tuberculosis.

In certain embodiments, the bacterial infection is tuberculosis.

Certain embodiments of the invention provide a compound of formula I, ora pharmaceutically acceptable salt thereof, or a compound of formula II,or a pharmaceutically acceptable salt thereof, for the prophylactic ortherapeutic treatment of a bacterial infection.

Certain embodiments of the invention provide a compound of formula I, ora pharmaceutically acceptable salt thereof, or a compound of formula II,or a pharmaceutically acceptable salt thereof, for use in medicaltreatment.

Certain embodiments of the invention provide the use of a compound offormula I, or a pharmaceutically acceptable salt thereof, or a compoundof formula II, or a pharmaceutically acceptable salt thereof, for thepreparation of a medicament for treating a bacterial infection in amammal.

Certain Definitions

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc.denote both straight and branched groups; but reference to an individualradical such as propyl embraces only the straight chain radical, abranched chain isomer such as isopropyl being specifically referred to.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase.

When a bond in a compound formula herein is drawn in anon-stereochemical manner (e.g. flat), the atom to which the bond isattached includes all stereochemical possibilities. When a bond in acompound formula herein is drawn in a defined stereochemical manner(e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understoodthat the atom to which the stereochemical bond is attached is enrichedin the absolute stereoisomer depicted unless otherwise noted. In oneembodiment, the compound may be at least 51% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 60% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 80% the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 90% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 95 theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 99% the absolute stereoisomer depicted.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl.

As used herein the term “Boc” refers to —C(═O)OC(CH₃)₃.

As used herein the term “salt” includes base addition, acid addition andquaternary salts. Compounds of the invention which are acidic can formsalts, including pharmaceutically acceptable salts, with bases such asalkali metal hydroxides, e.g. sodium and potassium hydroxides; alkalineearth metal hydroxides e.g. calcium, barium and magnesium hydroxides;with organic bases e.g. N-methyl-D-glucamine, cholinetris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine and the like. Those compounds which are basiccan form salts, including pharmaceutically acceptable salts withinorganic acids, e.g. with hydrohalic acids such as hydrochloric orhydrobromic acids, sulphuric acid, nitric acid or phosphoric acid andthe like, and with organic acids e.g. with acetic, tartaric, succinic,fumaric, maleic, malic, salicylic, citric, methanesulphonic,p-toluenesulphonic, benzoic, benzenesulfonic, glutamic, lactic, andmandelic acids and the like. For a review on suitable salts, seeHandbook of Pharmaceutical Salts: Properties, Selection, and Use byStahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

In cases where compounds are sufficiently basic or acidic, a salt of acompound of formula I or II can be useful as an intermediate forisolating or purifying a compound of formula I or II. Additionally,administration of a compound of formula I as a pharmaceuticallyacceptable acid or base salt may be appropriate. Pharmaceuticallyacceptable salts may be obtained using standard procedures well known inthe art.

Administration of Compounds of Formula I or II

As described herein, a compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a compound of formula II, or apharmaceutically acceptable salt thereof, can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I or II to the skin are known to theart; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria(U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) andWortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I or II can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Certain Methods for Preparing Compounds of Formula I or II

Generally, compounds of formula I and II, as well as syntheticintermediates that can be used for preparing compounds of formula I andII, can be prepared as illustrated in FIG. 2 and in the followingSchemes and Examples. It is understood that variable groups shown in theSchemes below (e.g. R¹, R², X and X₁) can represent the finalcorresponding groups present in a compound of formula I or II or thatthese groups can represent groups that can be converted to the finalcorresponding groups present in a compound of formula I or II at aconvenient point in a synthetic sequence. For example, in the Schemesbelow, the variable groups can contain one or more protecting groupsthat can be removed at a convenient point in a synthetic sequence toprovide the final corresponding groups in the compound of formula I orII. Processes for preparing compounds of formula I or II are provided asfurther embodiments.

In certain embodiments, X₁ in Scheme 5 above may be an aliphatic arm ofone or more methlylenes, which is boc-protected amino terminated oramine-terminated.

The antibiotic properties of a compound may be determined usingpharmacological models which are well known to the art, or using assaysdescribed in the Examples below (e.g., Test A).

The invention will now be illustrated by the following non-limitingExamples.

Example 1

Test A.

MIC/MBC studies were conducted according to a modified literatureprocedure (LaDow J E, et al. European Journal of Medicinal Chemistry. 46(2011) 4219-4226). In brief, stock amphiphile solutions were made in 1mL double-distilled water (0.1M C8 and C10; 0.02M C12), filtersterilized, and sonicated for five minutes. Stocks were then diluted100× in tryptic soy broth and vortexed. This broth was serially dilutedin fresh broth and 100 μL “doped” broth was transferred to 96-wellmicrotiter plate. 10⁶ Cfu/mL inoculums were prepared for E. coli and S.aureus. 100 μL inoculum was added to the wells. Plates were put on ashaker for 5 minutes to mix inoculum and broth. Plates were incubated at37° C. overnight. Wells were visually analyzed the next day by growth orno growth. After MICs were determined, aliquots were taken from eachwell, plated, and incubated to determine the MBC values for eachcompound. MBC values were defined as killing of ≥99.9% of the organisms,which corresponds to a 3-log reduction.

Data for the following representative compounds of formula (I) in Test Ais provided in Tables 1 and 2 below.

TABLE 1 MICs and MBCs (mM) of Amphiphiles* Amphiphile S. aureus E. coliC8 0.5 (0.5) 1 (1) C10 0.125 (0.25)  0.5 (0.5) C12 0.025 (0.05)  0.4(0.4) Streptomycin Sulfate <100 (<100) <100 (μg/mL) (<100) *Datarepresented in MIC (MBC) format.

TABLE 2 MICs and MBCs (μg/mL) of Amphiphiles* Amphiphile S. aureus E.coli C8 294 (294) 588 (588) C10 80.5 (171)  322 (322) C12 17.5 (35.0)280 (280) Streptomycin Sulfate <100 (<100) <100 (<100) *Data representedin MIC (MBC) format. MIC defined as inhibition of growth determined bythe unaided eye. MBC is defined as killing of ≥99.9% of the organisms,which corresponds to a 3-log reduction.These results demonstrate that compounds of the invention possessantibiotic properties.Materials and Methods

Cationic amphiphiles (8C, 10C and 12C) were synthesized according to aprocedure similar to the procedure illustrated in FIG. 2. The followingsynthesis is presented for a 1 g scale. Bromo-terminated fatty acidscontaining 8, 10, 12 carbons were stirred in concentrated ammoniumhydroxide solution (5, 50, or 100 mL with increasing carbon numbers) for1-2 days. Pure 1 was then isolated in vacuo. 1 (1 eq) was suspended in a1:1 mixture (9-18 mL each) of dioxane and 10% aqueous sodium carbonatesolution. The reaction mixture was warmed to 30° C. and additional wateradded if necessary to aid with stirring. Boc anhydride (1.1 eq) was thenadded and the reaction mixture heated to reflux temperatures (65° C.)and stirred overnight. The reaction mixture was concentrated in vacuo,reconstituted in 1N hydrochloric acid (HCl, 50 mL) and diethyl ether (15mL), and extracted with diethyl ether (4×50 mL). The combined etherlayers were washed with brine (50 mL), dried over magnesium sulfate, andconcentrated in vacuo to obtain 2. Next, 2 (2.2 eq),N,N-propyltartramide (1 eq), and catalytic dimethylaminopyridine (0.42eq) were dissolved in dichloromethane (DCM, 40-50 mL) and dimethylformamide (15-25 mL) under argon. Upon complete dissolution of allreagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4.2 eq) wasadded as a coupling reagent and the reaction stirred overnight. Thereaction mixture was concentrated in vacuo and reconstituted in DCM (50mL). This solution was washed with aqueous solutions of 10% potassiumbisulfite (3×50 mL), saturated sodium bicarbonate (3×50 mL) and brine(50 mL). The organic layer was then dried over MgSO₄ and 3 isolated invacuo. To obtain the final cationic products (4; 8C, 10C, and 12C), ananhydrous solution of 4M HCl in dioxane (40 eq) was cooled to 0° C.under argon. 3 (1 eq) was added and the reaction stirred at 0° C. underargon for 30 minutes. The reaction mixture was warmed to roomtemperature and stirred an additional three hours, after which thereaction mixture was concentrated in vacuo. The crude product wasdissolved in minimal methanol and pure product (4) precipitated withdiethyl ether (400 mL). 4 was isolated via centrifugation (3500 rpm, 5minutes) and the ether decanted.

8C. Off-white solid. ¹H-NMR (400 MHz, CD₃OD): δ 8.25 (b, 2H), 5.57 (s,2H), 3.15 (m, 4H), 2.92 (t, 4H), 2.48 (m, 4H), 1.64 (m, 8H), 1.51 (m,4H), 1.39 (b, 12H), 0.90 (t, 6H). ¹³C-NMR (500 MHz, CD₃OD): δ 172.65,167.62, 72.58, 41.18, 39.56, 33.24, 28.62, 27.29, 26.06, 24.36, 22.42,10.51. ESI-MS m/z: 517.9 [M+2], 516.8 [M+1], 258.0 [(M+2)/2].

10C. Off-white solid. ¹H-NMR (500 MHz, CD₃OD): δ 8.24 (b, 2H), 5.56 (s,2H), 3.14 (m, 4H), 2.91 (t, 4H), 2.46 (m, 4H), 1.63 (m, 8H), 1.50 (m,4H), 1.34 (b, 20H), 0.89 (t, 6H). ¹³C-NMR (500 MHz, CD₃OD): δ 172.68,167.54, 72.54, 41.06, 39.60, 33.29, 29.09, 29.08, 28.94, 28.88, 27.39,26.24, 24.49, 22.40, 10.50. ESI-MS m/z: 572.5 [M+2], 571.5 [M+1], 286.5[(M+2)/2].

12C. Off-white solid. ¹H-NMR (400 MHz, CD₃OD): δ 8.23 (b, 2H), 5.56 (s,2H), 3.14 (m, 4H), 2.91 (t, 4H), 2.45 (m, 4H), 1.63 (m, 8H), 1.50 (m,4H), 1.32 (b, 28H), 0.89 (t, 6H). ¹³C-NMR (500 MHz, CD₃OD): δ 172.68,167.55, 72.54, 41.07, 39.61, 33.33, 29.37, 29.34, 29.29, 29.22, 29.03,28.94, 27.41, 26.27, 24.54, 22.41, 10.50. ESI-MS m/z: 628.4 [M+2], 627.4(M+1), 314.3 [(M+2)/2].

Example 2 Biscationic Tartaric Acid-Based Amphiphiles: Charge LocationImpacts Antimicrobial Activity

As described herein, two series of cationic amphiphiles, termedbola-like and gemini-like amphiphiles, were synthesized with analogoushydrophobic-to-charge ratios but differing charge location and theirresulting antibacterial activity assessed. Bola-like amphiphilesexhibited preferential activity against two gram-positive bacteria, withactivity increasing with increasing hydrophobicity, whereas gemini-likeamphiphiles were active against both gram-positive and gram-negativebacteria, with activity decreasing with increasing hydrophobicity. Afteridentifying compounds from each amphiphile series (bola- andgemini-like), biophysical experiments indicated that both amphiphileswere membrane-active; notably, the gemini-like amphiphile (G7) exhibiteda strong dependence on electrostatic interactions for membraneinteraction. In contrast, the bola-like amphiphile (B11) exhibited areliance on both hydrophobic and electrostatic contributions. Theseresults demonstrate that charge location impacts cationic amphiphiles'antibacterial and membrane activity.

1. Introduction

In an effort to overcome current drawbacks of antimicrobial peptides(AMPs), many researchers have synthesized peptidomimetic compoundscontaining AMPs' key physicochemical properties, namely a net cationiccharge and amphiphilic structure. LaDow et al. developed a series ofaryl-based bicephalic amphiphiles (two cationic heads, one hydrophobictail, FIG. 5A) of varying hydrocarbon tail length and determined thatbicephalic compounds were more likely to be effective against bothgram-positive and gram-negative bacteria than conventional monocationicamphiphiles (LaDow, et al., European Journal of Medicinal Chemistry2011, 46, 4219). Building upon this work, Grenier et al. designed aseries of bipyridinium-based gemini amphiphiles (two cationic heads, twohydrophobic tails, FIG. 5A) that demonstrated improved antimicrobialactivity over bicephalic amphiphiles, with optimum activity occurring atintermediate hydrocarbon tail lengths (Grenier, et al., Bioorganic &Medicinal Chemistry Letters 2012, 22, 4055). Further, Mondal et al.conjugated cationic lysine residues onto glucose to generate bicephalicamphiphiles that may mimic peptide post-translational modifications ofAMPs (Mondal, et al., Carbohydrate Research 2011, 346, 588). In additionto investigating small molecule amphiphiles as antimicrobial agents,researchers have also studied oligomers (Liu, et al., AngewandteChemie-International Edition 2004, 43, 1158) and polymers (Scorciapino,et al., Biophys. J. 2012, 102, 1039; Paslay, et al., Biomacromolecules2012, 13, 2472; Gabriel, et al., Biomacromolecules 2008, 9, 2980;Palermo, E. F.; Vemparala, S.; Kuroda, K. Biomacromolecules 2012, 13,1632) in an attempt to develop potent bioactives. Paslay et al., forinstance, developed a series of poly(methacrylamide) (co)polymers whichdemonstrated increasing antimicrobial activity with increasing primaryamine content (Biomacromolecules 2012, 13, 2472).

In evaluating the diverse array of antimicrobial peptides andamphiphiles that have been developed, one trend becomes apparent:antimicrobial activity is largely influenced by a molecule'shydrophobic-to-charge ratio (Laverty, et al., International Journal ofMolecular Sciences 2011, 12, 6566; Grenier, et al., Bioorganic &Medicinal Chemistry Letters 2012, 22, 4055; LaDow, et al., EuropeanJournal of Medicinal Chemistry 2011, 46, 4219; Gabriel, et al.,Biomacromolecules 2008, 9, 2980; Palermo, et al., Biomacromolecules2012, 13, 1632). Very few studies, however, have compared amphiphilespossessing identical hydrophobic-to-charge ratios with varying chargelocations. Studies by LaDow et al. revealed that the spacing betweencationic charges on structurally similar bicephalic amphiphiles,containing the same hydrophobic-to-charge ratio, does influenceantimicrobial activity (LaDow, et al., European Journal of MedicinalChemistry 2011, 46, 4219). As described herein, the specific impact ofcharge location on cationic amphiphiles' antimicrobial activity wasexplored, while also delving into amphiphiles' specific membraneactivity.

To investigate this correlation, two series of sugar-based biscationicamphiphiles were synthesized with varying charge locations and varying,yet equivalent, hydrophobic-to-charge ratios. Each series had differingamphiphile architectures as a result of their charge location. Whereasone series more closely resembled gemini amphiphiles (two heads, twotails), which have been widely investigated for antimicrobialapplications (Grenier, et al., Bioorganic & Medicinal Chemistry Letters2012, 22, 4055; Zhang, et al., J. Polymer Chemistry 2012, 3, 907; IsabelMartin, et al., Colloids and Surfaces B-Biointerfaces 2014, 114, 247)the other was more bolaamphiphilic (two heads connected via one tail) innature (FIG. 5B). It was predicted that the gemini-like amphiphileswould exhibit improved antimicrobial activity compared to the bola-likeamphiphiles due to their more facially amphiphilic structure and thateach series' antimicrobial activity would increase with increasinghydrophobic-to-charge ratio due to enhanced hydrophobic interactions,leading to membrane permeabilization. Upon successful synthesis of allamphiphiles, their antimicrobial activity was assessed againstgram-negative and gram-positive bacteria. Certain compounds were furtherevaluated based on these results. Specifically, their interactions withmodel membranes via Langmuir monolayer techniques and isothermaltitration calorimetry (ITC) were measured.

2. Materials and Methods 2.1 Materials

All reagents and solvents were purchased from Sigma-Aldrich (Milwaukee,Wis.) and used as received unless otherwise noted. 1 N hydrochloric acid(HCl), concentrated ammonium hydroxide, deuterated methanol (CD₃OD),Petri dishes, and cotton swabs were purchased from Fisher Scientific(Fair Lawn, N.J.). Muller-Hinton agar and blank paper disks werepurchased from Becton Dickinson (Franklin Lakes, N.J.).1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) werepurchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.) and usedwithout further purification. N,N-dipropyl tartramide (PT) was preparedaccording to published procedures (Tounsi, et al., Journal of InorganicBiochemistry 2005, 99, 2423). For broth microdilution assays, bacterialstrains Escherichia coli ATCC®43895™, Pseudomonas aeruginosa ATCC®14213™, Listeria monocytogenes ATCC®49594™, and Staphylococcus aureusRosenbach ATCC® 29213™ were received from the American Tissue CultureCollection (ATCC, Manassass, Va., USA). The E. coli and S. aureusstrains were chosen because they are representative of gram-negative andgram-positive pathogens, respectively.

2.2 Characterization

Proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectrawere obtained using a Varian 400 or 500 MHz spectrometer. Samples weredissolved in deuterated chloroform (CDCl₃), dimethyl sulfoxide(DMSO-d₆), or CD₃OD using trimethylsilane or deuterated solvent (DMSO-d₆or CD₃OD) as an internal reference. Fourier transform infrared (FT-IR)spectra were obtained using a Thermo Scientific Nicolet iS10spectrophotometer equipped with OMNIC software. FT-IR samples wereeither pressed into potassium bromide (KBr) discs (1 wt % sample) orsolvent-cast onto sodium chloride plates; each spectrum was an averageof 32 scans. Molecular weights were determined using a ThermoQuestFinnigan LCQ-DUO system equipped with an atmospheric pressure ionization(API) source, a mass spectrometer (MS) detector, and the Xcalibur datasystem. Samples were prepared at a concentration of 10 μg/mL in methanol(MeOH) or 50:50 MeOH:dichloromethane (DCM).

2.3 Synthesis of Bola-Like Amphiphiles 2.3.1 Synthesis oftert-butyloxycarbonyl-(Boc-) Protected Alkanoic Acids (3) as Shown inScheme 6 Below

Following modified literature procedures (Orwig, et al., J Med. Chem.2009, 52, 1803; Amara, et al., Journal of the American Chemical Society2009, 131, 10610), bromo-terminated alkanoic acid (1, 3.62 mmol) waseither dissolved (1a) or suspended (1b-c) in concentrated ammoniumhydroxide (10-100 mL) and stirred for 24-48 h. Upon complete consumptionof starting material (monitored by thin layer chromatography, 75:25hexanes/ethyl acetate with acetic acid), the reaction mixture wasconcentrated in vacuo to isolate an amine-terminated alkanoic acidintermediate (2). The intermediate was then suspended in a 1:1 mixtureof dioxane and 10% sodium carbonate (14 mL each) and gently warmed to30° C. If necessary, additional water (5 mL) was added to improvestirring. Di-tert-butyl dicarbonate (3.98 mmol) was added and thereaction stirred under reflux temperatures (65° C.) overnight. Thereaction mixture was concentrated in vacuo and the resulting crudemixture reconstituted in 1 N HCl and diethyl ether and subsequentlyextracted with diethyl ether (4×80 mL). The combined organic layers werewashed with 1:1 brine/water (80 mL total), dried over magnesium sulfate(MgSO₄), and the product (3) isolated in vacuo.

8-Bocaminooctanoic Acid (3a).

Yield: 1.67 g, 78% (off-white solid). ¹H-NMR (500 MHz, CDCl₃): δ 4.56(br, 1H), 3.10 (m, 2H), 2.34 (t, 2H), 1.63 (m, 2H), 1.45 (m, 17H).¹³C-NMR (500 MHz, CDCl₃): δ 179.54, 156.29, 79.34, 40.77, 34.25, 30.16,29.17, 29.10, 28.64, 26.77, 24.83. IR (cm⁻¹, thin film from chloroform,CHCl₃): 3367 (NH), 1698 (C═O, acid and carbamate). ESI-MS m/z: 258.1[M−1].

10-Bocaminodecanoic Acid (3b).

Yield: 1.09 g, 97% (off-white solid). ¹H-NMR (500 MHz, CDCl₃): δ 4.59(br, 1H), 3.07 (m, 2H), 2.30 (t, 2H), 1.60 (m, 2H), 1.41 (m, 21H).¹³C-NMR (500 MHz, CDCl₃): δ 179.61, 156.30, 79.31, 40.81, 34.33, 30.18,29.49, 29.38, 29.32, 29.22, 28.62, 26.93, 24.91. IR (cm⁻¹, thin filmfrom CHCl₃): 3367 (NH), 1721 (C═O, acid), 1686 (C═O, carbamate). ESI-MSm/z: 286.1 [M−1].

12-Bocaminododecanoic Acid (3c).

Yield: 1.00 g, 97% (off-white solid). ¹H-NMR (500 MHz, CDCl₃): δ 4.61(br, 1H), 3.10 (m, 2H), 2.33 (t, 2H), 1.63 (m, 2H), 1.44 (m, 25H).¹³C-NMR (500 MHz, CDCl₃): δ 179.56, 156.29, 79.31, 40.85, 34.33, 30.21,29.66, 29.56, 29.46, 29.41, 29.26, 28.64, 28.45, 27.00, 24.94. IR (cm⁻¹,thin film from CHCl₃): 3368 (NH), 1722 (C═O, acid), 1686 (C═O,carbamate). ESI-MS m/z: 314.2 [M−1].

2.3.2 Synthesis of 2,3-bis(Boc-Protected Alkanoyl) PTs (4) as Shown inScheme 6 Below

PT (1.36 mmol), 3 (2.99 mmol), and catalytic dimethylaminopyridine(DMAP, 0.57 mmol) were dissolved in anhydrous DCM (27 mL) anddimethylformamide (DMF, 13 mL) under nitrogen. Upon completedissolution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 5.71mmol) was added and the reaction stirred overnight under nitrogen. Thereaction mixture was concentrated in vacuo, reconstituted in DCM, andwashed with aqueous solutions of 10% potassium bisulfite (KHSO₄, 3×80mL) and saturated sodium bicarbonate (NaHCO₃, 3×80 mL). The organiclayer was then washed with brine (80 mL), dried over MgSO₄, and theproduct (4) isolated in vacuo.

8-Bocaminooctanoyl PT (4a).

Yield: 1.25 g, 95% (pale-yellow solid). ¹H-NMR (500 MHz, CDCl₃): δ 6.27(br, 2H), 5.60 (s, 2H), 4.57 (br, 2H), 3.20 (m, 4H), 3.10 (m, 4H), 2.40(t, 4H), 1.63 (m, 4H), 1.44 (m, 38H), 0.91 (t, 6H). ¹³C-NMR (500 MHz,CDCl₃): δ 172.30, 166.38, 156.20, 79.22, 72.45, 41.44, 40.72, 34.00,30.18, 29.09, 29.03, 28.65, 26.76, 24.82, 22.86, 11.49. IR (cm⁻¹, thinfilm from CHCl₃): 3290 (NH), 1752 (C═O, ester), 1694 (C═O, carbamate),1655 (C═O, amide). ESI-MS m/z: 737.1 [M+23].

10-Bocaminodecanoyl PT (4b).

Yield: 0.81 g, 95% (pale-yellow solid). ¹H-NMR (500 MHz, CDCl₃): δ 6.24(br, 2H), 5.61 (s, 2H), 4.54 (br, 2H), 3.21 (m, 4H), 3.10 (m, 4H), 2.40(t, 4H), 1.62 (m, 4H), 1.51 (m, 46H), 0.91 (t, 6H). ¹³C-NMR (500 MHz,CDCl₃): δ 171.18, 165.18, 154.96, 78.05, 71.20, 40.17, 39.59, 32.86,29.04, 28.28, 28.17, 28.12, 27.96, 27.43, 25.73, 23.71, 21.64, 10.25. IR(cm⁻¹, thin film from CHCl₃): 3280 (NH), 1751 (C═O, ester), 1694 (C═O,carbamate), 1652 (C═O, amide). ESI-MS m/z: 793.2 [M+23].

12-Bocaminododecanoyl PT (4c).

Yield: 0.79 g, quantitative (pale-yellow solid). ¹H-NMR (400 MHz,CDCl₃): δ 6.35 (br, 2H), 5.61 (s, 2H), 4.54 (br, 2H), 3.19 (m, 4H), 3.09(m, 4H), 2.39 (t, 4H), 1.62 (m, 4H), 1.43 (m, 54H), 0.90 (t, 6H).¹³C-NMR (500 MHz, CDCl₃): δ 171.15, 165.20, 154.99, 77.93, 71.27, 40.20,39.61, 32.86, 29.06, 28.69, 28.49, 28.38, 28.26, 28.22, 28.03, 27.43,25.79, 23.74, 21.63, 10.27. IR (cm⁻¹, thin film from CHCl₃): 3281 (NH),1751 (C═O, ester), 1694 (C═O, carbamate), 1655 (C═O, amide). ESI-MS m/z:849.3 [M+23].

2.3.3 Synthesis of Bola-Like Amphiphiles (5) as Shown in Scheme 6 Below

Boc groups were deprotected following modified procedures (Han, et al.,Journal of Peptide Research 2001, 58, 338). In brief, HCl (4M indioxane, 50.78 mmol) was cooled to 0° C. under nitrogen, 4 added (1.27mmol), and the reaction stirred at 0° C. for 30 min. The reactionmixture was then warmed to room temperature, stirred an additional 3 h,and concentrated in vacuo. Crude product was dissolved in minimalmethanol (10 mL) and aliquots (1 mL) were added to ten 50 mL centrifugetubes containing diethyl ether (45 mL each), resulting in theprecipitation of 5. 5 was isolated via centrifugation (Hettich EBA 12,Beverly, Mass.; 1370×g, 5 min) and decanting the ether. Bola-likeamphiphiles will be referred to as Bx, where B denotes bola-like and xrefers to the number of methylenes in the acyl arms.

B7 (5a).

Yield: 0.55 g, 96% (off-white solid). ¹H-NMR (400 MHz, CD₃OD): δ 8.25(br, 2H), 5.57 (s, 2H), 3.15 (m, 4H), 2.92 (t, 4H), 2.48 (m, 4H), 1.64(m, 8H), 1.51 (m, 4H), 1.39 (br, 12H), 0.90 (t, 6H). ¹³C-NMR (500 MHz,CD₃OD): δ 172.65, 167.62, 72.58, 41.18, 39.56, 33.24, 28.62, 27.29,26.06, 24.36, 22.42, 10.51. IR (cm⁻¹, KBr): 3422 (NH), 1751 (C═O,ester), 1655 (C═O, amide). ESI-MS m/z: 258.0 [(M+2)/2].

B9 (5b).

Yield: 0.72 g, 95% (off-white solid). ¹H-NMR (500 MHz, CD₃OD): δ 8.24(br, 2H), 5.56 (s, 2H), 3.14 (m, 4H), 2.91 (t, 4H), 2.46 (m, 4H), 1.63(m, 8H), 1.50 (m, 4H), 1.34 (br, 20H), 0.89 (t, 6H). ¹³C-NMR (500 MHz,CD₃OD): δ 172.68, 167.54, 72.54, 41.06, 39.60, 33.29, 29.09, 29.08,28.94, 28.88, 27.39, 26.24, 24.49, 22.40, 10.50. IR (cm⁻¹, KBr): 3288(NH), 1749 (C═O, ester), 1670 (C═O, amide). ESI-MS m/z: 286.5 [(M+2)/2].

B11 (5c).

Yield: 0.86 g, 97% (off-white solid). ¹H-NMR (400 MHz, CD₃OD): δ 8.23(br, 2H), 5.56 (s, 2H), 3.14 (m, 4H), 2.91 (t, 4H), 2.45 (m, 4H), 1.63(m, 8H), 1.50 (m, 4H), 1.32 (br, 28H), 0.89 (t, 6H). ¹³C-NMR (500 MHz,CD₃OD): δ 172.68, 167.55, 72.54, 41.07, 39.61, 33.33, 29.37, 29.34,29.29, 29.22, 29.03, 28.94, 27.41, 26.27, 24.54, 22.41, 10.50. IR (cm⁻¹,KBr): 3288 (NH), 1744 (C═O, ester), 1668 (C═O, amide). ESI-MS m/z: 627.4[M+1].

2.4 Synthesis of Gemini-Like Amphiphiles 2.4.1 Synthesis of2-Bocaminoethyltartramide (2-Boc-AET) (7) as Shown in Scheme 7 Below

2-Boc-AET was prepared according to modified literature procedures(Tounsi, et al., Journal of Inorganic Biochemistry 2005, 99, 2423). Inbrief, dimethyl tartrate (6, 2.43 mmol) was dissolved in anhydroustetrahydrofuran (7.5 mL) under nitrogen. N-Boc-ethylenediamine (6.79mmol) was added and the reaction mixture stirred at 40° C. overnight.The crude reaction mixture was concentrated in vacuo and pure product(7) was triturated in diethyl ether (25 mL) and isolated via vacuumfiltration. To improve yields, the filtrate was reconcentrated in vacuo,triturated, and vacuum filtered to isolate additional pure product.Yield: 1.93 g, 91% (white solid). ¹H-NMR (400 MHz, DMSO-d₆): δ 7.79 (br,2H), 6.81 (br, 2H), 5.43 (d, 2H), 4.20 (d, 2H), 3.12 (m, 4H), 2.99 (m,4H), 1.36 (s, 18H). ¹³C-NMR (500 MHz, DMSO-d₆): δ 172.84, 156.35, 78.39,73.22, 40.43, 39.28, 28.92. IR (cm⁻¹, KBr): 3356 (NH), 1687 (C═O,carbamide), 1629 (C═O, amide). ESI-MS m/z: 457.2 [M+23].

2.4.2 Synthesis of 2,3-bis(alkanoyl) Boc-AET (8) as Shown in Scheme 7Below

Following methods similar to those described for the synthesis of 4,alkanoic acid (2.53 mmol), 7 (1.51 mmol), and DMAP (0.48 mmol) weredissolved in anhydrous DCM (50 mL) and anhydrous DMF (25 mL) undernitrogen. EDCI (4.83 mmol) was added, the reaction stirred overnight,and concentrated in vacuo. The crude mixture was reconstituted in DCM,washed with aqueous solutions of 10% KHSO₄ (3×80 mL), saturated NaHCO₃(3×80 mL), and brine (80 mL), dried over MgSO₄, and concentrated invacuo. This crude product was triturated in hexanes (160 mL) for 4 h andpure product (8) isolated via vacuum filtration.

Nonanoyl-Boc-AET (8a).

Yield: 0.76 g, 93% (white solid). ¹H-NMR (500 MHz, CDCl₃): δ 7.05 (br,2H), 5.58 (s, 2H), 5.18 (br, 2H), 3.30 (m, 8H), 2.45 (m, 4H), 1.63 (m,4H), 1.44 (18H), 1.27 (br, 20H), 0.88 (t, 6H). ¹³C-NMR (500 MHz, CDCl₃):δ 172.37, 167.04, 157.11, 79.94, 72.45, 41.21, 39.99, 34.06, 32.03,29.46, 29.36, 29.31, 28.62, 24.90, 22.85, 14.31. IR (cm⁻¹, thin filmfrom CHCl—₃): 3369 (NH), 1748 (C═O, ester), 1690 (C═O, carbamide), 1660(C═O, amide). ESI-MS m/z: 737.4 [M+23].

Undecanoyl-Boc-AET (8b).

Yield: 0.60 g, 84% (white solid). ¹H-NMR (500 MHz, CDCl₃): δ 7.13 (br,2H), 5.59 (s, 2H), 5.22 (br, 2H), 3.28 (m, 8H), 2.45 (m, 4H), 1.63 (m,4H), 1.44 (s, 18H), 1.26 (br, 28H), 0.88 (t, 6H). ¹³C-NMR (500 MHz,CDCl₃): δ 172.39, 167.08, 157.11, 79.90, 72.45, 41.16, 39.98, 34.04,32.11, 29.80, 29.72, 29.53, 29.51, 29.32, 28.61, 24.90, 22.89, 14.32. IR(cm⁻¹, thin film from CHCl₃): 3368 (NH), 1742 (C═O, ester), 1690 (C═O,carbamide), 1660 (C═O, amide). ESI-MS m/z: 793.4 [M+23].

Tridecanoyl-Boc-AET (8c).

Yield: 0.89 g, 94% (white solid). ¹H-NMR (500 MHz, CDCl₃): δ 7.07 (br,2H), 5.58 (s, 2H), 5.18 (br, 2H), 3.28 (m, 8H), 2.44 (m, 4H), 1.63 (m,4H), 1.44 (s, 18H), 1.26 (br, 36H), 0.88 (t, 6H). ¹³C-NMR (500 MHz,CDCl₃): δ 172.36, 167.06, 157.11, 79.93, 72.45, 41.22, 39.97, 34.05,32.14, 29.89, 29.87, 29.86, 29.73, 29.57, 29.53, 29.33, 28.62, 24.91,22.91, 14.33. IR (cm⁻¹, thin film from CHCl₃): 3367 (NH), 1740 (C═O,ester), 1689 (C═O, carbamide), 1659 (C═O, amide). ESI-MS m/z: 849.4[M+23].

2.4.3 Synthesis of Gemini-Like Amphiphiles (9) as Shown in Scheme 7Below

Boc groups were deprotected following the methods outlined for thesynthesis of 5. Briefly, HCl (4M in dioxane, 24.18 mmol) was cooled to0° C. under nitrogen, 8 added (0.60 mmol), and the reaction stirred at0° C. for 30 min. If necessary, additional anhydrous dioxane (3 mL) wasadded to improve stirring. The reaction mixture was warmed to roomtemperature, stirred an additional 3 h, and concentrated in vacuo. Crudeproduct was dissolved in minimal methanol (6 mL) and aliquots (1 mL)were added to six 50 mL centrifuge tubes containing diethyl ether (45 mLeach), resulting in the precipitation of 9. 9 was isolated viacentrifugation (Hettich EBA 12, Beverly, Mass.; 1370×g, 5 min) anddecanting the ether. Gemini-like amphiphiles will be referred to as Gx,where G denotes gemini-like and x refers to the number of methylenes inthe acyl arms.

G7 (9a).

Yield: 0.39 g, 95% (clear, off-white solid). ¹H-NMR (500 MHz, CD₃OD): δ8.64 (br, 2H), 5.58 (s, 2H), 3.52 (m, 4H), 3.09 (m, 4H), 2.49 (m, 4H),1.62 (m, 4H), 1.31 (br, 20H), 0.90 (t, 6H). ¹³C-NMR (500 MHz, CD₃OD): δ172.92, 168.94, 72.47, 39.46, 36.90, 33.42, 31.84, 29.25, 29.14, 29.00,24.61, 22.54, 13.28. IR (cm⁻¹, KBr): 3455 (NH), 1744 (C═O, ester), 1644(C═O, amide). ESI-MS m/z: 515.3 [M+1].

G9 (9b).

Yield: 0.42 g, quantitative (off-white solid). ¹H-NMR (500 MHz, CD₃OD):δ 8.62 (br, 2H), 5.57 (s, 2H), 3.50 (m, 4H), 3.08 (m, 4H), 2.47 (m, 4H),1.61 (m, 4H), 1.29 (br, 28H), 0.90 (t, 6H). ¹³C-NMR (500 MHz, CD₃OD): δ172.91, 168.95, 72.47, 39.46, 36.92, 33.43, 31.90, 29.57, 29.48, 29.30,29.01, 24.62, 22.56, 13.27. IR (cm⁻¹, KBr): 3435 (NH), 1739 (C═O,ester), 1652 (C═O, amide). ESI-MS m/z: 571.3 [M+1].

G11 (9c).

Yield: 0.40 g, 94% (white solid). ¹H-NMR (500 MHz, CD₃OD): δ 8.62 (br,2H), 5.57 (s, 2H), 3.52 (m, 4H), 3.09 (m, 4H), 2.49 (m, 4H), 1.62 (m,4H), 1.29 (br, 36H), 0.89 (t, 6H). ¹³C-NMR (500 MHz, CD₃OD): δ 172.90,168.95, 72.48, 39.47, 36.92, 33.43, 31.91, 29.64, 29.62, 29.61, 29.49,29.32, 29.02, 24.63, 22.56, 13.27. IR (cm⁻¹, KBr): 3448 (NH), 1744 (C═O,ester), 1641 (C═O, amide). ESI-MS m/z: 314.4 [(M+2)/2].

2.5 Antimicrobial Screening

Amphiphiles' antimicrobial activity against gram-negative (E. coli) andgram-positive (S. aureus) bacteria was first screened using the diskdiffusion method (Murray, et al., Manual of Clinical Microbiology; 7thed.; ASM Press: Washington, D C, 1999). Bacteria inocula were grownovernight in nutrient broth (EMD Chemicals, Gibbstown, N.J.) at 37° C.under shaking conditions to give a bacterial count of approximately 10⁸CFU/mL. Muller-Hinton agar was poured into sterile Petri dishes to athickness of 4 mm. The agar plate was then inoculated with the bacteriabroth culture using a sterile cotton swab. Separately, amphiphiles weredissolved in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES)buffer (10 mM, pH 7.4) at concentrations ranging from 0.8 mM to 100 mM.Sterile paper disks (6 mm diameter) were impregnated with 20 μL of testsolution and the disks placed onto the inoculated agar plates. Plateswere incubated at 37° C. for 20 h, after which zones of inhibition weremeasured with a ruler. HEPES buffer served as a negative control.

2.6 Broth Microdilution Assay

The broth microdilution method was modified from previous studies(LaDow, et al., European Journal of Medicinal Chemistry 2011, 46, 4219).Briefly, amphiphiles were serially diluted 2-fold in tryptic soy broth(TSB) and 100 μL aliquots of each dilution were transferred to a 96-wellmicrotiter plate in triplicate. S. aureus, L. monocytogenes, E. coli,and P. aeruginosa were grown on tryptic soy agar (TSA) at 37° C. for 24h, and sterile double-distilled water was inoculated with isolatedcolonies from these overnight plates. Inoculum concentration wasadjusted to 5×10⁶ CFU/mL with ultraviolet-visible (UV-Vis) spectroscopyat 600 nm. Aliquots (100 μL) were transferred to the 96-well microtiterplate to achieve a final concentration of 5×10⁵ CFU/well. Plates wereincubated at 37° C. for 24 h. The lowest amphiphile concentration thatyielded no visible growth was recorded as the minimum inhibitoryconcentration (MIC). Cetyltrimethylammonium bromide (CTAB), a cationicamphiphile, served as a positive control that could mimic the proposedbactericidal mechanism of the newly synthesized tartaric acid-basedcompounds. Sodium dodecyl sulfate (SDS), an anionic amphiphile, servedas an additional control, which was not expected to exhibit potentantimicrobial activity against the tested strains.

2.7 Langmuir Monolayer Studies

The ability of amphiphiles to penetrate lipid monolayers was analyzedusing a Langmuir surface balance equipped with a custom-builtmicrotrough from KSV-Nima (Biolin Scientific, Espoo, Finland). Lipidsolutions were prepared by dissolving DOPC, DOPG, or DOPC/DOPG (1:1 moleratio) in HPLC grade CHCl₃ (˜1.2 mg/mL total lipid). After rinsing withan ethanol/methanol mixture, the trough was filled with HEPES buffer andthe surface aspirated to remove surface-active particles. Using aHamilton syringe (Reno, Nev.), small aliquots of lipid solution wereapplied to the air/buffer interface to obtain varying initial surfacepressures ranging from approximately 17 mN/m to 38 mN/m. After solventevaporation and monolayer equilibration (at least 500 s), 5 μL of B11(Scheme 6) or G7 (Scheme 7) dissolved in HEPES buffer (5 mM initialamphiphile) was injected into the aqueous subphase via a side port toavoid puncturing the monolayer and the surface pressure increasemonitored over time. Data were collected and processed using KSV Nimaand Origin software.

2.8 Isothermal Titration Calorimetry

High sensitivity isothermal titration calorimetry (MicroCal VP-ITC,Malvern Instruments, Westborough, Mass.) was used to assess theenergetics of amphiphile interactions with lipid vesicles. Largeunilamellar vesicles (LUVs) comprised of DOPC or DOPC:DOPG (1:1 moleratio) were prepared following a published extrusion method (Zhang, etal., Journal of Biological Chemistry 2014, 289, 11584). In brief, driedlipid films (pure DOPC or DOPC:DOPG 1:1 mole ratio) were hydrated withHEPES buffer, subject to 5 freeze-thaw cycles, and extruded through 100nm polycarbonate filters 10 times using a nitrogen-driven device (LipexBiomembranes, Vancouver, BC, Canada).

The ITC sample cell (˜1.4 mL) was filled with solutions of 25 μM B11(Scheme 6) or G7 (Scheme 7) dissolved in HEPES buffer, and the referencecell was filled with the same buffer. The syringe (250 μL) was filledwith LUV dispersions containing 5 mM total lipid. All solutions weredegassed for 10 min prior to each experiment. Upon system equilibrationand a 1 μL pre-injection, 5 L aliquots were injected into the samplecell every 11 min for the first 4 injections, after which time aliquotswere injected in 8-min intervals. Data were collected and processedusing proprietary software from MicroCal. All experiments were performedat least in triplicate. Titrations of LUVs into buffer were conducted asnegative controls and subtracted from experimental data.

3. Results and Discussion 3.1 Amphiphile Synthesis

To explore the impact of charge location on antimicrobial activity, twoseries of cationic amphiphiles (gemini- and bola-like) were synthesizedwith equivalent hydrophobic-to-charge ratios (FIG. 5B). Both seriesemployed tartaric acid, an inexpensive naturally occurring compoundproduced in fruits (Wu, et al., Biomacromolecules 2008, 9, 2921), as abackbone that could provide two distinct chemical moieties for furthermodification. By altering the charge location on these tartaricacid-based molecules, two structurally diverse amphiphile series weredeveloped.

Bola-like amphiphiles resulted when cationic charges were incorporatedat the terminal ends of hydrophobic acyl arms (Scheme 6). This serieswas synthesized by first reacting bromo-containing alkanoic acids (1)with concentrated ammonium hydroxide to generate amine-terminatedalkanoic acid intermediates (2). The amine-terminated alkanoic acidswere then Boc-protected (3) using di-tert-butyl dicarbonate andsubsequently conjugated to a PT backbone using carbodiimide coupling togenerate 4. Following successful acylation, 4 was deprotected using HClin dioxane to generate the final bola-like amphiphiles (5) as chloridesalts. All amphiphiles' and intermediates' chemical structures wereconfirmed via NMR and FT-IR spectroscopies and mass spectrometry.

A series of gemini-like amphiphiles was synthesized by incorporatingcationic charges at the tartaric acid backbone. These amphiphilespossessed analogous molecular weights, chemical moieties (e.g., numberof amine moieties or methylene units), and hydrophobic-to-charge ratiosas the bola-like amphiphiles, differing only in their charge location(FIG. 5B). To synthesize these molecules, dimethyl tartrate was firstreacted with N-Boc-ethylenediamine via an aminolysis reaction togenerate 7 (Scheme 7). 7 was then acylated with alkanoic acids ofvarying hydrophobic chain lengths using carbodiimide coupling and theBoc protecting groups removed using HCl in dioxane to generate the finalamphiphile structures (9). Successful synthesis of the gemini-likeamphiphiles and their intermediates was confirmed as described above.

3.2 Antimicrobial Activity

Antimicrobial activity was first screened using the disk diffusionmethod, a qualitative assay which indicated that all amphiphiles exceptG11—the most hydrophobic gemini-like amphiphile—exhibited activityagainst S. aureus, L. monocytogenes, E. coli, or P. aeruginosa in themillimolar range (4-100 mM, FIGS. 9 & 10). While this method isexcellent for screening, it is not always suitable for the assessment ofhydrophobic compounds as they diffuse more slowly through the agar andmay not accurately depict bioactivity (Klancnik, et al., J. Microbiol.Methods 2010, 81, 121). Consequently, a broth microdilution assay wascarried out to quantitatively assess amphiphile activity. Amphiphileswere incubated with S. aureus or E. coli in TSB; the lowest amphiphileconcentrations that yielded no visible bacterial growth were taken asthe MIC values. With the exception of G11, whose antibacterialassessment was hampered by poor aqueous solubility, all amphiphilesexhibited MICs within the low micromolar to low millimolar range (Table3).

TABLE 3 MICs (μM) of Amphiphiles S. aureus L. monocytogenes E. coli P.aeruginosa Amphiphile (G+) (G+) (G−) (G−) B7 500 500 1000 1000 B9 125125 500 250 B11  25* 12.5* 100   50* G7 62.5* 62.5* 62.5* 125 G9 500 250125 250 G11 >200 >200 >200 >200 CTAB 4 8 32 16 SDS 1000 2000 >2000 >2000*Cationic amphiphile treatments possessing MIC values lower than 50μg/mL

In comparing amphiphiles' antibacterial activity, it became apparentthat the hydrophobic-to-charge ratio, which was investigated by varyingthe number of methylene units present in amphiphiles' hydrophobicdomains, significantly influenced amphiphile bioactivity (Table 3).Within the bola-like series (B7, B9, B11), amphiphiles exhibitedincreasing antibacterial activity as the number of methylene unitsincreased, with B11 demonstrating the highest potency againstgram-positive (MIC: 25 and 12.5 μM against S. aureus and L.monocytogenes, respectively) and gram-negative (MIC: 100 and 50 μMagainst E. coli and P. aeruginosa, respectively) bacteria. These resultsalign with previous findings, which indicate increasing acyl chainlengths can result in enhanced bioactivity so long as solubility is notdrastically diminished (LaDow, et al., European Journal of MedicinalChemistry 2011, 46, 4219). Furthermore, a recent study by Palermo et al.indicated that antimicrobial activity increases as the spacer lengthbetween ammonium ions and a methacrylate polymer backbone increases(Palermo, et al., Biomacromolecules 2012, 13, 1632). Given that themethylenes of the bola-like amphiphiles' acyl arms are analogous to suchspacer units, these compounds may behave similarly, with longer acylarms allowing for enhanced membrane penetration and increasedbioactivity. In contrast to the trends noted for the bola-likeamphiphiles, gemini-like amphiphiles (G7, G9, G11) exhibited decreasedantimicrobial activity with increasing acyl chain length. Previousstudies have indicated that amphiphiles' with poor solubility exhibitdecreased antibacterial activity, as they are incapable of reaching thebacterial membrane (Tew, et al., Accounts of chemical research 2010, 43,30; Grenier, et al., Bioorganic & Medicinal Chemistry Letters 2012, 22,4055; LaDow, et al., European Journal of Medicinal Chemistry 2011, 46,4219). Upon increasing gemini-like amphiphiles' acyl chain length toG11, the amphiphile could not dissolve above 200 μM in TSB. It isplausible that gemini-like amphiphiles' decreased solubility in TSBcompromised their antibacterial activity. Although solubility effectsmay have influenced the gemini-like amphiphile series, G7 exhibited highefficacy against S. aureus, L. monocytogenes, and E. coli (MICs: 63 μM).As compounds that exhibit MIC values ≤50 μg/mL are commonly consideredantimicrobial (Scorciapino, et al., Biophys. J. 2012, 102, 1039), brothmicrodilution studies enabled the identification of two compounds—B11and G7—whose micromolar MIC values correspond to values ranging from 8.7and 37.0 μg/mL (denoted by asterisks in Table 3).

When the two series' effects on gram-positive and gram-negative bacteriawere compared, varying trends emerged. Bola-like amphiphiles exhibitedhigher activity against gram-positive organisms S. aureus and L.monocytogenes, which may result from bola amphiphiles' tendency topenetrate membranes without causing membrane disruption (O'Toole, etal., Cornea 2012, 31, 810). Given that gram-negative bacteria contain anadditional outer membrane, this potential mechanism of action could haverendered bola-like amphiphiles less active against gram-negativebacteria. Furthermore, as gram-positive and gram-negative bacteriapossess different types and ratios of lipids within their cellmembranes, it is plausible that the bola-like amphiphiles' enhancedactivity against S. aureus and L. monocytogenes results frominteractions with specific lipid components. Gemini-like amphiphilesexhibited no definitive trends against the different bacteria classes;however, G7's high activity against S. aureus, L. monocytogenes, and E.coli indicates that gemini-like amphiphiles may possess broader activityagainst both gram-positive and gram-negative bacteria. Given that thebola-like and gemini-like amphiphiles were influenced differently bytheir hydrophobic-to-charge ratios and exhibited varying activitiesagainst gram-positive and gram-negative bacteria, it is plausible thatthe two series act via different bactericidal mechanisms. Future studiesinvestigating a more expansive series of gram-positive and gram-negativebacteria may further elucidate the potential relationship betweenbacteria classes and antibacterial activity.

3.3 Biophysical Assessment

Two amphiphiles—B11 and G7—were selected for further experimentationbased on their antibacterial activity. As many AMPs interact withbacterial membranes (Park, et al., International Journal of MolecularSciences 2011, 12, 5971; Laverty, et al., International Journal ofMolecular Sciences 2011, 12, 6566), it was predicted that these twocompounds may also interact with bacterial membranes as part of theirbactericidal mechanisms. To this end, Langmuir monolayer assays and ITCexperiments were conducted to ascertain how the compounds interact withmodel membrane systems. Given that bola-like and gemini-like amphiphilesexhibited different trends in antibacterial activity, experiments wereperformed to further understand whether B11 and G7 would exhibitdifferent interactions with model membranes.

3.3.1 Langmuir Monolayer Studies: B11 and G7 can PreferentiallyPenetrate Anionic Biomembranes

Langmuir monolayer techniques were employed to understandamphiphile/lipid interactions. Within these studies, neutral DOPCmonolayers served to mimic eukaryotic membranes, whereas anionic DOPG orDOPC:DOPG (1:1 mol ratio) monolayers served to mimic bacterial membranesand elucidate the influence of charge on membrane interactions.Monolayers of varying initial surface pressures were spread at theair/buffer interface and the surface pressure increase monitored uponinjection of either B11 or G7 into the aqueous subphase. By plotting thechange in surface pressure as a function of initial surface pressure,the x-intercept—corresponding to the amphiphiles' maximum insertionpressure (MIP)—was extrapolated (Calvez, et al., Biochimie 2009, 91,718). MIP values denote the maximum pressure at which insertion into themonolayer is favorable and provide a quantitative means to compareamphiphile/lipid interactions. As MIP values higher than 30-35 mN/m areindicative of biomembrane penetration (Calvez, et al., Biochimie 2009,91, 718), this methodology provides insight into B11 and G7 interactionswith eukaryotic and/or bacterial membranes.

B11 and G7 exhibited no significant incorporation into neutral DOPCmonolayers (FIG. 6), with negligible changes in surface pressure and nolinear regression with increasing initial surface pressures. Incontrast, both amphiphiles interacted with anionic monolayers (FIG. 6),exhibiting a surface pressure increase, which decreased with higherinitial surface pressures (FIG. 7). This enhanced membrane activity inthe presence of anionic lipids has been previously reported(Scorciapino, et al., Biophys. J 2012, 102, 1039) and may indicate thatthe amphiphiles behave similarly to cationic AMPs, which initiallyinteract with bacterial membranes via electrostatic interactions(Laverty, et al., International Journal of Molecular Sciences 2011, 12,6566; Brogden, K. A. Nature Reviews Microbiology 2005, 3, 238). In DOPGand DOPC:DOPG monolayers, B11 exhibited MIP values of 40 and 42 mN/m,respectively, whereas G7 exhibited MIP values of 46 and 52 mN/m,respectively (FIG. 7). In general, G7's higher MIP values indicateenhanced interactions with anionic monolayers. As all MIP values weregreater than the biomembrane lateral pressure and comparable to MIPvalues of known AMPs (Calvez, et al., Biochimie 2009, 91, 718), it isexpected that both B11 and G7 are capable of intercalating withinanionic bacterial membranes, suggesting that these amphiphiles maybehave similarly to AMPs and target the bacterial membrane as part oftheir bactericidal mechanism. In comparing amphiphile interactions withthe two different anionic lipid systems, both amphiphiles exhibitedhigher MIP values in the presence of DOPC:DOPG monolayer mixtures. Thisphenomenon could result from DOPC's smaller head group area(Kleinschmidt, et al., Biophys. J. 2002, 83, 994) enabling a morefavorable insertion of amphiphiles into the lipid monolayer.

3.3.2 Langmuir Monolayer Studies Suggest Electrostatic Contributions inMembrane Interaction Differ for B11 and G7

In addition to extrapolating MIP values, a second parameter thatprovides useful information for analyzing membrane interaction is themaximum surface pressure increase measured during Langmuir monolayerstudies (Calvez, et al., Biochimie 2009, 91, 718). Through comparing theamphiphiles' maximum surface pressure increase in the presence of bothDOPG and DOPC:DOPG, we could better understand the influence ofmonolayer charge on amphiphile adsorption. This value is typicallyobtained by comparing adsorption curves with the same initial surfacepressure; however, both amphiphiles exhibited a plateau in maximumsurface pressure increase at lower initial surface pressures, likely dueto their equilibrium with the bulk aqueous phase. Consequently, thelowest initial surface pressures plotted in FIG. 7, correspond to themaximum surface increase for a given amphiphile/lipid system.

G7 exhibits a maximum surface pressure increase of 24 mN/m in thepresence of pure DOPG, which decreases to 12 mN/m in the presence ofDOPC:DOPG (FIG. 7B). This dependence of maximum surface pressureincrease on the mole fraction of anionic lipid has been previouslyreported (Kennedy, et al., Biochemistry 1997, 36, 13579) and indicatesan electrostatic contribution in membrane binding. B11 also exhibits adecrease in maximum surface pressure increase when changing the lipidsystem from DOPG to DOPC:DOPG, yet to a smaller extent (20 mN/m to 15mN/m, FIG. 7A) than G7; this result reflects a lesser dependence onelectrostatic interactions. These results are further emphasized in FIG.6; B11 behaves similarly in the presence of both DOPG and DOPC:DOPG,whereas G7 exhibits a drastic decrease in surface pressure increase uponchanging the lipid system from DOPG to DOPC:DOPG. The notable differencein electrostatic contribution suggests that B11 relies on a combinationof electrostatic and hydrophobic interactions to elicit bacterial death,whereas G7's bactericidal mechanism may be largely driven byelectrostatic interactions. B11's reliance on both hydrophobic andelectrostatic interactions for monolayer intercalation, could suggestthat the bola-like compounds are drawn to the bacterial membrane via aninitial electrostatic interaction followed by intercalation into thehydrophobic membrane interior via hydrophobic interactions. G7's strongelectrostatic interaction with anionic monolayers suggests thatgemini-like compounds may interact predominantly with anionic componentsof bacterial membranes, including PG headgroups, lipopolysaccharide(LPS, found in gram-negative bacteria), and lipoteichoic acids (LTA,found in gram-positive bacteria). A primarily electrostatic mechanism ofaction could be hampered by increasing hydrophobic content, potentiallyresulting in gemini-like amphiphiles' decreased activity with increasinghydrophobic-to-charge ratio.

3.3.3 ITC Studies: B11 and G7 Operate Via Different BactericidalMechanisms

While Langmuir monolayer studies provided valuable insight intoamphiphile's interactions with biomembranes, ITC was used to investigateamphiphiles' interactions with bilayers, a more relevant model membranesystem. LUVs comprised of pure DOPC or DOPC:DOPG (1:1 mol ratio) wereprepared to mimic eukaryotic and bacterial membranes, respectively;these LUVs were titrated into a sample cell containing amphiphilesolution (i.e., B11 or G7 dissolved in HEPES buffer). Both B11 and G7exhibited no interactions with neutral DOPC LUVs, evidenced bynegligible heat signals during the titration (see, FIGS. 11 and 12). Aseukaryotic membranes also exhibit a net neutral charge, these resultsmay indicate that both amphiphiles would interact minimally witheukaryotic cells, a correlation that has been previously depicted byEpand et al. (J. Mol. Biol. 2008, 379, 38). In investigating anionicLUVs (i.e., DOPC:DOPG), both amphiphiles exhibited binding interactions,and the heats associated with these binding interactions generallydecreased as the titrations progressed. As LUVs were added into thetitration cell amphiphiles would bind to LUVs, leaving less amphiphilesavailable for binding and resulting in smaller heat signals insubsequent LUV injections until all amphiphiles were removed from thebulk solution (Seelig, J. Biochim. Biophys. Acta-Rev. Biomembr. 1997,1331, 103; Breukink, et al., Biochemistry 2000, 39, 10247; Domingues, etal., Langmuir 2013, 29, 8609; Binder, et al., Biophys. J. 2003, 85,982). B11 exhibited endothermic binding interactions, indicated by apositive enthalpy change (FIG. 8A). Such binding interactions oftenresult from the displacement of counterions or water molecules as aresult of the hydrophobic effect (Seelig, J. Biochim. Biophys. Acta-Rev.Biomembr. 1997, 1331, 103; Gabriel, et al., Langmuir 2008, 24, 12489;Seelig, J. Biochim. Biophys. Acta-Biomembr. 2004, 1666, 40), suggestingB11's hydrophobic domain may penetrate into the hydrophobic membraneinterior of the anionic LUVs and that binding is largely influenced viahydrophobic interactions. As B11 did not interact with DOPC (i.e.,neutral) LUVs yet did interact with DOPC:DOPG (i.e., anionic) LUVs, itwas predicted that an initial electrostatic interaction occurred.Although electrostatic binding results in exothermic heat signals, it isplausible B11's stronger dependence on the hydrophobic effect resultedin the observed positive enthalpy change (Livne, et al., Chemistry &Biology 2009, 16, 1250).

In contrast to B11, G7 exhibited a negative enthalpy change underidentical conditions (FIG. 8B), which suggests an exothermic,electrostatic interaction between G7 and anionic LUVs (Epand, et al., JMol. Biol. 2008, 379, 38). This exothermic interaction supports Langmuirmonolayer data, which indicated that G7's membrane insertion activityinvolved a larger electrostatic contribution than that of B11.

The diverging energetics of binding indicate that B11 and G7 may act viadifferent bactericidal mechanisms. Gemini-like amphiphiles demonstratedactivity against both gram-positive and gram-negative organisms. As G7exhibits electrostatic binding interactions with anionic LUVs, theseamphiphiles may interact favorably with the negatively charged lipidcomponents of gram-positive and gram-negative bacteria (e.g., lipidheadgroups, LPS, or LTA), enabling specific activity against bacteria.For instance, G7 may interact with LPS on the outer membrane of E. coli,potentially neutralizing LPS or displacing divalent cations associatedwith LPS and ultimately distorting the outer membrane (Park, et al.,International Journal of Molecular Sciences 2011, 12, 5971; Laverty, etal., International Journal of Molecular Sciences 2011, 12, 6566). Thiselectrostatic interaction may have been hampered upon increasingcompounds' hydrophobic-to-charge ratio, resulting in decreased activity.After this initial electrostatic interaction, gemini-like compoundslikely insert their hydrophobic tails into the hydrophobic membraneinterior; however, this interaction was not observed during biophysicalstudies. In contrast, bola-like amphiphiles exhibited preferentialactivity against gram-positive bacteria with B11 demonstratingendothermic binding interactions with anionic LUVs, indicative of theentropically driven hydrophobic effect. These molecules likely rely onan initial electrostatic interaction, with the negatively chargedpeptidoglycan matrix of gram-positive bacteria, followed byintercalation into the membrane's hydrophobic domain, potentiallyadopting a U-shape or membrane-spanning conformation. This reliance onhydrophobic interactions could explain why bola-like amphiphilesexhibited enhanced activity upon increasing their hydrophobic-to-chargeratio. Modeling studies are currently underway to understand thisconformation. Over time, this intercalation may result in membranedestabilization through various potential mechanisms, such as membranethinning or pore formation (Park, et al., International Journal ofMolecular Sciences 2011, 12, 5971; Laverty, et al., InternationalJournal of Molecular Sciences 2011, 12, 6566; Brogden, K. A. NatureReviews Microbiology 2005, 3, 238; Mondal, et al., Carbohydrate Research2011, 346, 588). In summary, the antimicrobial studies in conjunctionwith biophysical experiments described herein indicate the significantinfluence of charge location on amphiphile activity.

4. Conclusions

Bola-like and gemini-like amphiphiles were synthesized to understand thespecific influence of charge location on antibacterial activity.Bola-like amphiphiles exhibited increased activity with increasinghydrophobic-to-charge ratios, likely resulting from a combination ofboth hydrophobic and electrostatic interactions with the bacterialmembranes. Gemini-like amphiphiles demonstrated a different trend, withantibacterial activity increasing as hydrophobic-to-charge ratiosdecreased. This phenomenon may have resulted from the decreasedsolubility of more hydrophobic gemini-like amphiphiles or fromgemini-like amphiphiles relying primarily on electrostatic interactionsin their bactericidal mechanism. Additionally, both amphiphilesexhibited differences in bioactivity against the tested gram-positivebacteria and gram-negative bacteria, further suggesting that the twoamphiphile series possess different bactericidal mechanisms and mayinteract with different components of bacteria membranes. These studiesreveal that, in addition to the hydrophobic-to-charge ratio, chargelocation significantly modulates cationic amphiphiles' antibacterialactivity and bactericidal mechanism. Through understanding thisinfluence of charge location, antimicrobial agents could be designed totarget different bacteria types and/or membrane structures.

Example 3 Antimicrobial Amphiphiles with Enhanced Activity

The antimicrobial activity of the G7, G9 and B11 amphiphiles against avariety of bacterial species was evaluated using MIC studies.Specifically, Standard CLIS/NCCLS broth microdilution assays wereutilized for both BSL2 and BSL3 organisms in a 96-well format((CLSI/NCCLS) CaLSI. Methods for antimicrobial susceptibility testing ofaerobic bacteria: Approved standard. Wayne, Pa.: CLIS, 2007;(CLSI/NCCLS) CaLSI. Methods for dilution antimicrobial susceptibilitytests for bacteria that grow aerobically; Approved standard. Wayne, Pa.:CLIS, 2009). The compounds were diluted at 32 mg/mL to 0.125 mg/mL in2-fold dilutions performed in triplicate. The time course of killing wasestablished by evaluating viability after exposure to compound after 18hours. The MIC is defined as the lowest concentration of compound thatconfers a no-growth phenotype as noted by the naked eye. These studiesdemonstrate broad spectrum activity and impact of the amphiphiles (Table4).

TABLE 4 MIC Values (ug/mL) G7 G9 B11 Vero Cell Cytotoxicity >50 50 50 A.baumannii >50 >50 >50 B. cereus 6.25 6.25 50 B. cepacia >50 >50 >50 B.neotomae 3.125 3.125 25 E. faecium 6.25 6.25 25 K. pneumoniae 50 >50 >50L. pneumophilia 12.5 25 >50 P. aeruginosa 25 >50 >50 S. aureus 6.25 6.2525 Y. pseudotuberculosis 12.5 6.25 >50 S. epidermidis 12.5 6.25 25

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A compound of formula II:

wherein: R¹ is a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b); R² is a (C₁-C₆)alkyl that is substituted with one or moreNR_(a)R_(b); each X is independently (C₄-C₁₂)alkyl; R_(a) is eachindependently H or (C₁-C₆)alkyl; R_(b) is each independently H,(C₁-C₆)alkyl or —C(═NH)NH₂; and n is 1, 2, 3, 4, 5, 6, 7, or 8; or asalt thereof.
 2. The compound or salt of claim 1, wherein R¹ is methyl,ethyl, propyl, isopropyl, butyl, isobutyl, or sec-butyl, substitutedwith one or more NR_(a)R_(b).
 3. The compound or salt of claim 1,wherein R² is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, orsec-butyl, substituted with one or more NR_(a)R_(b).
 4. The compound orsalt of claim 1, wherein each X is independently (C₈)alkyl.
 5. Thecompound or salt of claim 1, wherein each X is independently (C₉)alkyl.6. The compound or salt of claim 1, wherein each X is independently(C₁₀)alkyl.
 7. The compound or salt of claim 1, wherein each X isindependently (C₁₁)alkyl.
 8. The compound or salt of claim 1, whereineach X is independently (C₁₂)alkyl.
 9. The compound or salt of claim 1,wherein n is
 2. 10. The compound or salt of claim 1, which is a compoundof formula (IIc):

or a salt thereof.
 11. The compound or salt of claim 1, which isselected from the group consisting of:

or a salt thereof.
 12. The salt of claim 1, which is selected from thegroup consisting of:


13. A pharmaceutical composition comprising a compound as described inclaim 1, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 14. The compound or salt of claim10, wherein each X is independently (C₇)alkyl, (C₉)alkyl or (C₁₁)alkyl.15. The compound or salt of claim 1, which is:

or a salt thereof.